A stem cell is defined as a self-renewing primitive cell that can divide indefinitely (i.e. it is immortal) and can develop into functional, differentiated cells. Differentiated cells are of value for the treatment of degenerative disease or injury by transplantation, or as in vitro model systems for pharmaceutical or toxicology screening. A limitation of differentiated cells however, is their limited capacity to proliferate in culture.
Stem cells can be derived from embryonic, fetal and adult tissue, with embryo stem cells exhibiting the greatest capacity for immortal self-renewal (i.e. expansion), and pluripotency to form differentiated cells. However, the immortal character of stem cells when undifferentiated can result in the formation of tumours if transplanted into a living organism. This is a critical problem for the use of stem cells in the treatment of degenerative diseases or injuries such as for example Parkinson's disease, diabetes, spinal cord repair, or bone repair, etc. Therefore what is needed are methods to control the immortality of cells without compromising their useful proliferative capacity and their lineage potential. That is, what is needed is to re-create a proliferative but mortal population of cells, similar to mesenchyme, which forms during embryo gastrulation, whose lineage potential is retained and controlled by local microenvironments.
Immortal stem cell self-renewal can be accomplished in vitro by growth of cells on either differentiated helper/feeder cells or under feeder-free conditions on extracellular matrix substrates by supplementation of media with defined growth factors or cytokines. For example, human embryo stem cells (hESCs) can be self-renewed by growth in feeder-free culture system consisting of growth on Matrigel™ (MG), a commercially available matrix derived from mouse tumours containing mostly laminin, collagen IV, and heparin sulfate proteoglycan) (Xu et al., 2001). Growth of hESCs on this matrix still requires prior conditioning of culture medium by feeder cells and supplementation with bFGF. Under these conditions laminin could also support hESC self-renewal, whereas other extracellular matrix (ECM) molecules such as Fibronectin and Collagen could not (Xu et al., 2001). However, hESC interactions with the ECM may be growth factor dependent since Fibronectin can support hESC self-renewal provided that the medium is further supplemented with transforming growth factor β1 (TGFβ1) and Leukemia inhibitory Factor (LIF) in addition to bFGF (Amit M et al., 2004).
The first ES cell lines derived from either non-human primates or humans were obtained using medium containing bovine fetal serum and mitotically inactivated mouse embryonic fibroblasts (MEF) as supporting feeder layers (1, 2). Prior to this, derivation of hES without feeders resulted in uncontrolled cell differentiation with successive passages (3). Recently, it has become possible to maintain hES in an undifferentiated state by relying instead on MEF conditioned medium and cultivation on the extra-cellular matrix (ECM) molecule laminin alone or with collagen, and heparan sulfate in a matrigel matrix (4). Medium conditioned by mouse embryonic or human adult fibroblasts or epithelial cells immortalized with hTERT cannot prevent hES from differentiating, despite growth on ECM substrates (4). Thus, one or more factors secreted by feeder cells, such as mouse embryo-derived fibroblasts, uniquely contribute to hES cell maintenance on ECM substrates.
Stem cell differentiation can be mediated in vitro by a variety of methods involving culture of cells that are adherant to a substrate or in suspension. For example:                Indirect embryoid body mediated differentiation of cell representative of all germinal lineages (endoderm, ectoderm, mesoderm) (Keller, 1995; Itskovitz-Eldor et al., 2000; Conley et al., 2004)        Directed differentiation of neural cells by suspension culture of neurospheres (Zhang et al., 1998; Uchida et al., 2000; Schumacher et al., 2003)        Differentiation of cells grown on differentiated helper/feeder cells (Richards et al., 2002; Amit et al., 2003; Amit et al., 2004)        Differentiation of cells grown on specified extracellular matrix molecules (Xu et al., 2001; Rosler et al., 2004)        
The problem with all of these strategies is that they favour either a heterogeneous or single population of terminally differentiated cell phenotypes whose proliferative capacity is lost. What is needed is a method for the controlled differentiation of an intermediate progenitor population with proliferative capacity that retains its potential to yield a single lineage in response to specific environmental cues. In animal development cells that conceptually possess such properties evolve during the process of gastrulation, which results in the formation of three primary germ layers, ectoderm, mesoderm, and endoderm. Of these layers, mesenchymal cells derived from the mesoderm layer have the unique capacity to proliferate and migrate to specific sites, in developing organisms referred to as limb buds, at which they become terminally differentiated and normally give rise to muscle, skeletal (i.e. bone), blood, vascular and urogenital systems and connective tissue, specifically osteoblasts, chondroblasts, adipocytes, fibroblasts, cardiomyoctes and skeletal myoblasts.
In the adult, mesenchymal cells can be recovered from bone marrow. These are referred to as mesenchymal stem cells because they can be cultured ex-vivo for a limited number of passages and be differentiated at the single cell level into mesodermal cell types as described. When introduced in vivo bone mesenchymal cells can differentiate into the same array of cell types, as well as cells with characteristics of cells outside the mesoderm, including endothelium, neuroectoderm, and endoderm (reviewed in Verfaillie et al. 2002). The formation of mesenchyme during gastrulation in the embryo, or in bone marrow during development or in the adult organism, is a complex process mediated by the interaction of a broad range of soluble and insoluble factors, which include amongst others glycosominoglycans. The complexity of this process is such however, that it is unlikely that a single factor could yield the unique properties of mesenchymal cells.
Glycosaminoglycans (GAGs) are unbranched chains composed of repeating disaccharide units. These disaccharide units always contain an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which in most cases is sulfated, with the second sugar usually being a uronic acid (glucuronic or iduronic). GAGs are highly negatively charged because of the presence of carboxyl or sulfate groups on most of their sugar residues. As such they are strongly hydrophilic. GAGs tend to adopt highly extended conformations and form matrices that are space filling and resistant to compressive forces. Four main groups of GAGs have been distinguished by their sugar residues, the type of linkage between these residues, and the number and location of sulfate groups. They include: (1) hyaluronan, (2) chondroitin sulphate and dermatan sulfate, (3) heparan sulfate and heparin, and (4) keratan sulfate.
Hyaluronan (also called hyaluronic acid or hyaluronate) is the simplest of GAGs. It consists of a regular repeating sequence of non-sulfated disaccharide units, specifically N-acetylglucosamine and glucuronic acid. Its molecular weight can range from 400 daltons (the disaccharide) to over a million daltons. It is found in variable amounts in all tissues, such as the skin, cartilage, and eye, and in most if not all fluids in adult animals. It is especially abundant in early embryos. Space created by hyaluronan, and indeed GAGs in general, permit it to play a role in cell migration, cell attachment, during wound repair, organogenesis, immune cell adhesion, activation of intracellular signalling, as well as tumour metastasis. These roles are mediated by specific protein and proteoglycan binding to Hyaluronan. Thus, in articular cartilage, hyaluronan and aggrecan form large aggregates important for the function of cartilage (Hardingham and Muir, 1972). Furthermore, cell motility and immune cell adhesion is mediated by the cell surface receptor RHAMM (Receptor for Hyaluronan-Mediated Motility; Hardwick et al., 1992) and CD44 (Jalkenan et al., 1987; Miyake et al., 1990).
Hyaluronan is synthesized directly at the inner membrane of the cell surface with the growing polymer extruded through the membrane to the outside of the cell as it is being synthesized. Synthesis is mediated by a single protein enzyme, hyaluronan synthetase (HAS) whose gene family consists of at least 3 members. By contrast other GAGs are synthesized inside the cell in the Golgi apparatus, possibly in association with some core protein, and then released by exocytosis. Hyaluronan degradation in vertebrate tissues in vivo is mediated by hyaluronidase, and exoglycosidases that remove sugars sequentially Mammalian-type hyaluronidases have both hydrolytic and transglycosidase activities and can degrade hyaluronan and chondroitin as well as, to a small extent, dermatan sulfate.
Human ES cells will have several distinct roles in medicine, for which it is important that lines are available from a variety of different genotypes. Ideally it should be practicable to obtain ES cells with a new specific genotype by their derivation from embryos produced by nuclear transfer. In addition to their potential use in cell therapy, such lines will create new opportunities for studies of drug toxicology, for drug discovery and studies of diseases that have a genetic component, but not necessarily a result of inappropriate function of a single gene. Although human embryonic stem (hES) cell lines show great promise for the development of cell-based therapies to replace damaged tissues, the currently available lines may be of little utility for this purpose. This is because of their provenance. To be used a source of tissue for transplantation in the future, hES cells will have to be derived and maintained under far more stringent conditions of 1) informed consent from the donor and 2) bio-safety, than hitherto observed. Central to bio-safety issues is the capacity to derive and maintain hES cells or their derivatives under defined and pathogen free conditions. This requirement is severely constrained by the dependence of existing protocols on animal feeder cells and undefined factors provided by feeder cells, conditioned medium, or exogenously supplied serum.
It has now been found that Hyaluronan can surprisingly assist in the differentiation of pluripotent stem cells to derive a mortal multi-lineage progenitor cell population which resemble adult mesenchymal stem cells.